1. Field of the Invention
This invention, in general, relates to a method for protecting moist stratified squamous epithelia against damage from exposure to noxious luminal substances.
In particular, this invention relates to:
I) the identification of a mechanism for protecting moist stratified squamous epithelia against damage from exposure to luminal substances such as hydrochloric acid (HCl) and N-acetylcysteine. This is based on the recognition that some of the noxious effects of luminal substances on moist stratified squamous epithelia are a consequence of their ability to increase the permeability across the intercellular junctions (paracellular pathway), and that moist stratified squamous epithelia can be protected from the noxious effect of luminal exposure to these substances by stabilizing their intercellular junctions so as to block the increase in permeability through this route; and
II) the identification of 3 chemical reactive groups and three classes of chemical compounds derived from them that provide protection to moist stratified squamous epithelia against damage from luminal HCl or N-acetylcysteine by stabilizing their intercellular junctions so as to block the increase in paracellular permeability that normally accompanies such exposure.
More specifically this invention relates to:
A) the identification of a new mechanism for protection of esophageal epithelia against acid injury and that is by stabilizing their intercellular junctions so as to block the increase in paracellular permeability that occurs with exposure to luminal acid; the ability to protect by preventing the increase in paracellular permeability represents a new mechanism of "cytoprotection", cytoprotection by definition meaning a method of protecting against acid injury to tissue without inhibiting gastric acid secretion or altering luminal acidity,
B) the identification that the mechanism described in (A) also applies to protection of esophageal epithelia against damage from luminal N-acetylcysteine and other noxious luminal substances that primarily damage by increasing permeability through the paracellular pathway,
C) the identification that the mechanism described in (A) also applies to protection of buccal and other moist stratified squamous epithelia against damage from luminal HCl,
D) the identification of three chemical reactive groups and three classes of chemical compounds derived from them that with topical application protect esophageal epithelia against damage from luminal HCI by stabilizing their intercellular junctions so as to block the HCl-induced increase in paracellular permeability.
The three reactive groups are:
a) X-SO.sub.3.sup.-, where X represents an oxygen or carbon linkage covalently or ionically bound to an organic or inorganic molecule, and the tetrahedral-shaped divalent oxy-anionic groups: PA1 b) XO.sub.4.sup.= and PA1 c) X.sub.2 O.sub.7.sup.=, where X represents an element from group VIb of the periodic table or sulfur from group VIa covalently or ionically bound to an organic or inorganic molecule. PA1 a) the sulfonates, e.g. 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS), 8-anilino-naphthalene-1-sulfonate (ANS), dinitrodisulfonic acid stilbene (DNDS), sulfonazo III, 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), bromphenol blue, PA1 b) the sulfate esters, e.g. sucrose octasulfate, dextran sulfate, and PA1 c) the tetrahedral-shaped divalent oxy-anions, e.g. sodium chromate, sodium dichromate, sodium molybdate, sodium tungstate, sodium sulfate, PA1 a) X-SO.sub.3.sup.-, where X represents an oxygen or carbon linkage covalently or ionically bound to an organic or inorganic molecule, and the tetrahedral-shaped divalent oxy-anionic groups: PA1 b) XO.sub.4.sup.= and PA1 c) X.sub.2 O.sub.7.sup.=, where X represents an element from group VIb of the periodic table or sulfur from group VIa covalently or ionically bound to an organic or inorganic molecule. PA1 a) the sulfonates, e.g. 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS), 8-anilino-naphthalene-1-sulfonate (ANS), dinitro-disulfonic acid stilbene (DNDS), sulfonazo III (sodium salt of 3,6-bis-(o-sulfophenylazo)-4,5-dihydroxy 2.7-naphthalenedisulfonic acid), 4,4'-diisothiocyano-2,2'stilbene disulfonic (DIDS), bromphenol blue, PA1 b) the sulfate esters, e.g. sucrose octasulfate, dextran sulfate, and PA1 c) the tetrahedral-shaped divalent oxy-anions, e.g. sodium chromate, sodium dichromate, sodium molybdate, sodium tungstate, sodium sulfate,
The three classes of chemical compounds derived from them are:
E) the identification that the reactive groups and classes of compounds referred to in (D) also protect esophageal epithelia against damage from luminal N-acetylcysteine and other luminal agents that damage by increasing paracellular permeability,
F) the identification that the reactive groups and classes of compounds referred to in (D) also protect buccal epithelia against damage from exposure to HCl and other luminal agents that damage by increasing paracellular permeability,
G) the identification that the reactive groups and classes of compounds referred to in (D), if shown to be non-toxic to humans, can be used orally as a treatment to prevent reflux esophagitis or its symptoms (e.g., heartburn),
H) the establishment of a technique using the Ussing chamber and voltage clamp for identifying agents capable of protecting esophageal, buccal and other moist stratified squamous epithelia against luminal HCl, N-acetylcysteine and other noxious luminal agents that damage by increasing the permeability through the paracellular pathway.
2. Background Information
Reflux esophagitis is a chronic disease that results from repeated and prolonged contact of esophageal epithelium with gastric acid (Richter J. E., Castell DO. Gastroesophageal Reflux: Pathogenesis, Diagnosis, and Therapy. Ann Intern Med 1982; 97-103). This represents one of the most common illnesses in humans as attested to by the fact that there is almost universal appreciation of its characteristic symptom, "heartburn". It has been reported that 7% of the U.S. population experience heartburn on a daily basis and that 36% experience it at least once per month (Nebel TO, Fornes MF, Castell DO: Symptomatic gastroesophageal reflux: Incidence and precipitating factors. Am J Dig Dis 1976; 21:953). In general terms reflux esophagitis and its major symptom, heartburn, develop as a result of the chemical damaging effects of gastric acid on the esophageal epithelium, and progression of this damaging action can lead to esophageal ulceration with bleeding, esophageal obstruction due to stricture formation and the development of a Barrett's esophagus (i.e. replacement of the squamous epithelial lining by a metaplastic columnar epithelium), the latter a premalignant lesion. For these reasons reflux esophagitis is a serious disease.
There are two general approaches to treatment of reflux esophagitis. The first and thus far most successful is to reduce gastric acidity. This is usually accomplished by inhibiting HCl secretion with either agents like cimetidine that block the parietal cell H.sub.2 -receptor or with agents like omeprazole that block the parietal cell enzyme H-K ATPase. Although the former have been only moderately successful, the latter which have recently been marketed reportedly heal up to 95% of patients with reflux disease. However the relapse rates when therapy is stopped are high for both types of agents, and that for omeprazole is 80% within six months of stopping therapy. Further although patients may respond to retreatment or be prevented from relapse by continuing the drug, there is concern about the safety of omeprazole when used long term. This is because rats treated with omeprazole for long periods have developed gastric carcinoid tumors and these are believed to be secondary to the rebound hypergastrinemia associated with potent acid suppression. For this reason there remains a need for therapeutic agents that can prevent relapse of reflux symptoms and esophagitis following treatment with the more potent acid-suppressing agents.
The second approach to treating reflux esophagitis involves using agents that enhance one of the intrinsic defenses of the esophagus. For example bethanecol and metoclopromide have been used because of their abilities to increase the contractility of the lower esophageal sphincter (LES). This may theoretically be beneficial because the LES is the major barrier to reflux in humans. In practice however these agents have not been very effective.
Another defense mechanism that may be enhanced is that of the tissue's intrinsic resistance to acid digestion, and agents that increase "tissue resistance" without inhibiting gastric acid secretion or luminal buffering have been referred to as "cytoprotective". The inventors' laboratory has been interested in identifying compounds that protect esophagus against acid damage by a cytoprotective action on the epithelium. Such agents, the inventors found, can be identified in vitro by mounting esophageal epithelia in Ussing chambers hooked to voltage clamps and showing that an agent prevents the reduction in the tissue's electrical resistance (R) upon exposure to luminal HCl. The ability of an agent to block the HCl-induced decline in R confers protection on esophageal epithelia because the inventors had previously shown that the HCl-induced decline in R, reflecting an increase in tissue permeability through the intercellular junctions (paracellular pathway), precedes the development of cell necrosis (Orlando, R. C., D. W. Powell, and C. N. Carney. Pathophysiology of acute acid injury in rabbit esophageal epithelium. J. Clin. Invest, 68:286-293, 1981 and Orlando, R. C., J. C. Bryson, and D. W. Powell. Mechanisms of H injury in rabbit esophageal epithelium. Am J. Physiol, 246(Gastrointest. Liver Physiol. 9): G718-G724, 1984). Additional work by the inventors indicates that cell necrosis occurs after the increase in paracellular permeability because it is the latter that allows hydrogen ions to enter the epithelium at a sufficient rate to overcome intercellular buffering by serosal bicarbonate (Tobey, N. A., D. W. Powell, V. J. Schreiner and R. C. Orlando. Serosal bicarbonate protects against acid injury to rabbit esophagus. Gastroenterology 96:1466-77, 1989). The inability to buffer the increased amounts of hydrogen ions eventually leads to cell death by acidifying the region adjacent to the basolateral cell membrane, the basolateral cell membrane unlike the apical cell membrane being unable to tolerate even modest lowering of bathing solution pH without damage to the cell (Tobey NA, Orlando RC. Comparative sensitivity of rabbit esophageal epithelium to serosal versus luminal acid. Gastroenterology 1989; 96:A512).
Sucralfate is a cytoprotective drug developed by Chugai in Japan and marketed in the U.S. by Marion Laboratories. It has been used for treatment of duodenal ulcer in the U.S. and more recently has been studied as a possible treatment for reflux esophagitis in humans (Weiss W., Brunne H., Buttner G. R., et al. Treatment of reflux esophagitis with sucralfate. Dtsch Med Wochenschr 1983; 108:1706). Although the efficacy of sucralfate in humans with reflux esophagitis has not been dramatic, the inventors experiments with sucralfate in esophageal epithelia mounted in Ussing chambers suggested that it contained a potent cytoprotective compound, and that this compound was sucrose octasulfate (Orlando RC, N. A. Turjman, N. A. Tobey, V. J. Schreiner, D. W. Powell. Mucosal protection by sucralfate and its components in acid-exposed rabbit esophagus. Gastroenterology 1987; 93:352-61). Additional experiments also lead the inventors to recognize that the essential component responsible for the cytoprotective property of both sucrose octasulfate and sucralfate was the presence of sulfate ions within the molecules (Tobey, N. A., R. C. Orlando, V. J. Schreiner, D. W. Powell. Cytoprotective effect of sulfate ions in acid-exposed rabbit esophagus. Am J Physiol 1986; 251:G 866-869). Also noteworthy was that protection by sucrose octasulfate and sulfate ions in vitro using the Ussing chamber-voltage clamp technique were validated by showing that these same compounds exert protection in vitro in resected specimens from human esophagus (un published observations) and in vivo using as a model the acid-perfused rabbit esophagus (Orlando RC et al. Gastroenterology 1987; 93:352-61 and Tobey NA et al. AM J Physiol 1986; 251:G866-869).
Although the mechanism by which inorganic sulfate ions protected against acid damage to esophageal epithelium was unknown and initially considered to be unique (Tobey NA et al. Am J Physiol 1986; 251:G866-869), a chance occurrence lead us to identify that 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS) also protected Ussing-chambered esophageal epithelium against luminal HCl injury in similar manner and at doses far lower than sulfate ions (Tobey NA, Schreiner V. J., Orlando R. C. Protection by SITS in acid-exposed rabbit esophagus. Gastroenterology 1988; 94:A461). Interestingly the inventors learned from the literature that sulfonates like SITS have a strong affinity for the same membrane receptor as sulfate ions and as such inhibit sulfate transport in rat kidney cells (Fritzsch, G., G. Rumrich, K. J. Ullrich. Anion transport through the contraluminal cell membrane of renal proximal tubule. The influence of hydrophobicity and molecular charge distribution on the inhibitory activity of organic anions. Biochimica et Biophysica Acta 1989; 978:249-256 and K. J. Ullrich, G. Rumrich, S. Kloss. Contraluminal sulfate transport in the proximal tubule of the rat kidney. II Specificity: sulfate-ester, sulfonates and amino sulfonates. Pflugers Arch 1985; 404:293-299). This suggested to the inventors the possibility that sulfonates may protect esophageal epithelia against acid injury by binding to the same receptor in this tissue as sulfate ions. Similar reasoning was applied to the possibility that tetrahedral-shaped divalent oxy-anions of elements from group VIb of the periodic table (e.g. chromate) might protect against acid injury to esophageal epithelia when it was learned from the literature that these compounds also bind to sulfate receptors and inhibit sulfate transport in vesicles from placental epithelial cells (Boyd, CAR, D. B. Shennan. Sulphate transport into vesicles prepared from human placental brush border membranes: inhibition by trace element oxides. J Physiol 1986; 379: 367-376). The present invention is based in large part on confirmation of this hypothesis--that is, that binding to a similar site in esophageal epithelium as sulfate ions confers on these additional agents the same protective properties against acid injury observed with sulfate treatment (see summary of invention below). Further the present invention also is broader based in that the protective mechanisms and the identified protective agents also apply to protection of esophageal epithelium against other luminal damaging agents (e.g. N-acetylcysteine) and to protection of other moist stratified squamous epithelia, e.g. buccal epithelium, against injury from luminal HCl.